Different Regulation of Physiological and Tumor Angiogenesis in Zebrafish by Protein Kinase D1 (PKD1)

Protein kinase D isoenzymes (PKDs, Prkds) are serine threonine kinases that belong to the CAMK superfamily. PKD1 is expressed in endothelial cells and is a major mediator of biological responses downstream of the VEGFRs that are relevant for angiogenesis such as endothelial cell migration, proliferation and tubulogenesis in vitro. PKDs also play a critical role in tumor development and progression, including tumor angiogenesis. However, given the plethora of signaling modules that drive angiogenesis, the precise role of PKD1 in both physiological and tumor angiogenesis in vivo has not been worked out so far. This study aimed at dissecting the contribution of PKD1 to physiological blood vessel formation, PKD1 was found to be widely expressed during zebrafish development. As far as physiological angiogenesis was concerned, morpholino-based silencing of PKD1 expression moderately reduced the formation of the intersomitic vessels and the dorsal longitudinal anastomotic vessel in tg(fli1:EGFP) zebrafish. In addition, silencing of PKD1 resulted in reduced formation of the parachordal lymphangioblasts that serves as a precursor for the developing thoracic duct. Interestingly, tumor angiogenesis was completely abolished in PKD1 morphants using the zebrafish/tumor xenograft angiogenesis assay. Our data in zebrafish demonstrate that PKD1 contributes to the regulation of physiological angiogenesis and lymphangiogenesis during zebrafish development and is essential for tumor angiogenesis.     

Motoneurons are essential for vascular pathfinding

The neural and vascular systems share common guidance cues that have direct and independent signaling effects on nerves and endothelial cells. Here, we show that zebrafish Netrin 1a directs Dcc-mediated axon guidance of motoneurons and that this neural guidance function is essential for lymphangiogenesis. Specifically, Netrin 1a secreted by the muscle pioneers at the horizontal myoseptum (HMS) is required for the sprouting of dcc-expressing rostral primary motoneuron (RoP) axons and neighboring axons along the HMS, adjacent to the future trajectory of the parachordal chain (PAC). These axons are required for the formation of the PAC and, subsequently, the thoracic duct. The failure to form the PAC in netrin 1a or dcc morphants is phenocopied by laser ablation of motoneurons and is rescued both by cellular transplants and overexpression of dcc mRNA. These results provide a definitive example of the requirement of axons in endothelial guidance leading to the parallel patterning of nerves and vessels in vivo.     

Live imaging of lymphatic development in the zebrafish

The lymphatic system has become the subject of great interest in recent years because of its important role in normal and pathological processes. Progress in understanding the origins and early development of this system, however, has been hampered by difficulties in observing lymphatic cells in vivo and in performing defined genetic and experimental manipulation of the lymphatic system in currently available model organisms. Here, we show that the optically clear developing zebrafish provides a useful model for imaging and studying lymphatic development, with a lymphatic system that shares many of the morphological, molecular and functional characteristics of the lymphatic vessels found in other vertebrates. Using two-photon time-lapse imaging of transgenic zebrafish, we trace the migration and lineage of individual cells incorporating into the lymphatic endothelium. Our results show lymphatic endothelial cells of the thoracic duct arise from primitive veins through a novel and unexpected pathway.     

Smooth muscle-endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation

Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels--smooth muscle and endothelial cells--and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function.     

An In Vivo Method to Quantify Lymphangiogenesis in Zebrafish

Background

Lymphangiogenesis is a highly regulated process involved in the pathogenesis of disease. Current in vivo models to assess lymphangiogenesis are largely unphysiologic. The zebrafish is a powerful model system for studying development, due to its rapid growth and transparency during early stages of life. Identification of a network of trunk lymphatic capillaries in zebrafish provides an opportunity to quantify lymphatic growth in vivo.

Methods and Results

Late-phase microangiography was used to detect trunk lymphatic capillaries in zebrafish 2- and 3-days post-fertilization. Using this approach, real-time changes in lymphatic capillary development were measured in response to modulators of lymphangiogenesis. Recombinant human vascular endothelial growth factor (VEGF)-C added directly to the zebrafish aqueous environment as well as human endothelial and mouse melanoma cell transplantation resulted in increased lymphatic capillary growth, while morpholino-based knockdown of vegfc and chemical inhibitors of lymphangiogenesis added to the aqueous environment resulted in decreased lymphatic capillary growth.

Conclusion

Lymphatic capillaries in embryonic and larval zebrafish can be quantified using late-phase microangiography. Human activators and small molecule inhibitors of lymphangiogenesis, as well as transplanted human endothelial and mouse melanoma cells, alter lymphatic capillary development in zebrafish. The ability to rapidly quantify changes in lymphatic growth under physiologic conditions will allow for broad screening of lymphangiogenesis modulators, as well as help define cellular roles and elucidate pathways of lymphatic development.     

A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy

During mammalian development, a subpopulation of endothelial cells in the cardinal vein (CV) expresses lymphatic‐specific genes and subsequently develops into the first lymphatic structures, collectively termed as lymph sacs. Budding, sprouting and ballooning of lymphatic endothelial cells (LECs) have been proposed to underlie the emergence of LECs from the CV, but the exact mechanisms of lymph vessel formation remain poorly understood. Applying selective plane illumination‐based ultramicroscopy to entire wholemount‐immunostained mouse embryos, we visualized the complete developing vascular system with cellular resolution. Here, we report emergence of the earliest detectable LECs as strings of loosely connected cells between the CV and superficial venous plexus. Subsequent aggregation of LECs resulted in formation of two distinct, previously unidentified lymphatic structures, the dorsal peripheral longitudinal lymphatic vessel (PLLV) and the ventral primordial thoracic duct (pTD), which at later stages formed a direct contact with the CV. Providing new insights into their function, we found vascular endothelial growth factor C (VEGF‐C) and the matrix component CCBE1 indispensable for LEC budding and migration. Altogether, we present a significantly more detailed view and novel model of early lymphatic development.     

Inhibition of Plk1 induces mitotic infidelity and embryonic growth defects in developing zebrafish embryos

Polo-like kinase 1 (Plk1) is central to cell division. Here, we report that Plk1 is critical for mitosis in the embryonic development of zebrafish. Using a combination of several cell biology tools, including single-cell live imaging applied to whole embryos, we show that Plk1 is essential for progression into mitosis during embryonic development. Plk1 morphant cells displayed mitotic infidelity, such as abnormal centrosomes, irregular spindle assembly, hypercondensed chromosomes, and a failure of chromosome arm separation. Consequently, depletion of Plk1 resulted in mitotic arrest and finally death by 6 days post-fertilization. In comparison, Plk2 or Plk3 morphant embryos did not display any significant abnormalities. Treatment of embryos with the Plk1 inhibitor, BI 2536, caused a block in mitosis, which was more severe when used to treat plk1 morphants. Finally, using an assay to rescue the Plk1 morphant phenotype, we found that the kinase domain and PBD domains are both necessary for Plk1 function in zebrafish development. Our studies demonstrate that Plk1 is required for embryonic proliferation because its activity is crucial for mitotic integrity. Furthermore, our study suggests that zebrafish will be an efficient and economical in vivo system for the validation of anti-mitotic drugs.     

lyve1 expression reveals novel lymphatic vessels and new mechanisms for lymphatic vessel development in zebrafish

We have generated novel transgenic lines that brightly mark the lymphatic system of zebrafish using the lyve1 promoter. Facilitated by these new transgenic lines, we generated a map of zebrafish lymphatic development up to 15 days post-fertilisation and discovered three previously uncharacterised lymphatic vessel networks: the facial lymphatics, the lateral lymphatics and the intestinal lymphatics. We show that a facial lymphatic vessel, termed the lateral facial lymphatic, develops through a novel developmental mechanism, which initially involves vessel growth through a single vascular sprout followed by the recruitment of lymphangioblasts to the vascular tip. Unlike the lymphangioblasts that form the thoracic duct, the lymphangioblasts that contribute to the lateral facial lymphatic vessel originate from a number of different blood vessels. Our work highlights the additional complexity of lymphatic vessel development in the zebrafish that may increase its versatility as a model of lymphangiogenesis.     

Vascular endothelial growth factor receptor-3 activity is modulated by its association with caveolin-1 on endothelial membrane

Vascular endothelial growth factor receptor-3 (VEGFR-3) is constitutively expressed in lymphatic vessels and transiently in endothelial cells of blood vessels during angiogenesis. Here we report that VEGFR-3 localizes in the caveolae membrane of endothelial cells and co-immunoprecipitates with caveolin-1. Caveolin-1 silencing or its depletion from the cell membrane by cholesterol increases VEGFR-3 autophosphorylation, suggesting that caveolin acts as a negative regulator of VEGFR-3 activity. Receptor activation induces caveolin-1 phosphorylation on tyrosine residues including tyrosine 14. Cell treatment with Src or Abl inhibitors PP2 or STI571, prior to receptor stimulation, affects caveolin-1 phosphorylation without affecting receptor autophosphorylation, suggesting that both Src and Abl are involved in VEGFR-3-dependent caveolin-1 phosphorylation. Caveolin-1 phosphorylation in Src/Fyn/Yes knockout cells demonstrated that Abl phosphorylates caveolin-1 independently from Src family members. These results suggest a functional interaction between VEGFR-3 and caveolin-1 to modulate endothelial cell activation during angiogenesis.     

Hypoxia/Notch signaling in primary culture of rat lymphatic endothelial cells

We have developed an improved intralumenal digestion method to get a long-term primary culture of rat lymphatic endothelial cells (rLECs) that maintained their original phenotypes. rLECs in vitro under hypoxia retained their original lymphatic properties observed in the thoracic duct. Blocking Notch signal with a gamma-secretase inhibitor and transfection of a cDNA expressing a dominant negative form of Delta1 indicated that Notch signal downregulated VEGFR-2 under hypoxia and inhibited cell migration. These findings indicated that Notch signal was still operative in mature lymphatic endothelial cells in response to the oxygen concentration.     

Development of the thoracic duct in the prenatal period of human ontogeny

In 40 series of histological sections performed in human embryos and prefetuses from 4 up to 20 weeks of development, as well as in 20 corpses of fetuses and stillborns, it has been stated that the anlage of the thoracic duct appear in 6-7-week-old fetuses as lymphatic clefts surrounded with mesenchymal cells that are situated near large veins in the areas of the most active morphogenesis. Connecting with each other, the clefts form the jugular and retroperitoneal lymph sacs and a well branching network of canals. From the latter, on the 7th-8th week of development a plexus of lymph vessels appear, and later on (on the 8th-9th week)--bilaterally situating trunks of the thoracic duct. Further development of the thoracic duct is connected with the lymph nodes formation, their germs appear on the 9th-10th week along the course of the left trunk, as well as along the ductal branches and anastomoses. The formation of the lymph nodes results in reduction of some trunks and plexuses of the thoracic duct. Owing to this, its form in 14-15-week-old prefetuses resembles the one in newborns. Disturbances in the formation processes of the lymph nodes along the course of the reducing ductal areas, as well as their formation along the course of its main trunk can result in various structural variants of the thoracic duct in children and grown-up persons. Histogenesis of the thoracic duct wall and formation of the lymph nodes are not completed by birth.     

Co-expression of α9β1 integrin and VEGF-D confers lymphatic metastatic ability to a human breast cancer cell line MDA-MB-468LN

Introduction and Objectives

Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice.

Results

A comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or α9 in 468LN cells.

Conclusion

Differential capacity for VEGF-D production and α9β1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype.     

Modeling lymphangiogenesis in a three-dimensional culture system

A lack of appropriate in vitro models of three-dimensional lymph vessel growth hampers the study of lymphangiogenesis. We developed a lymphatic ring assay--a potent, reproducible and quantifiable three-dimensional culture system for lymphatic endothelial cells that reproduces spreading of endothelial cells from a pre-existing vessel, cell proliferation, migration and differentiation into capillaries. In the assay, mouse thoracic duct fragments are embedded in a collagen gel, leading to the formation of lumen-containing lymphatic capillaries, which we assessed by electron microscopy and immunostaining. We developed a computerized method to quantify the lymphatic network. By applying this model to gene-deficient mice, we found evidence for involvement of the matrix metalloproteinase, MMP-2, in lymphangiogenesis. The lymphatic ring assay bridges the gap between two-dimensional in vitro models and in vivo models of lymphangiogenesis, can be used to exploit the potential of existing transgenic mouse models, and rapidly identify regulators of lymphangiogenesis.     

Suppression of AGO2 by miR-132 as a determinant of miRNA-mediated silencing in human primary endothelial cells

The abundance of miR-132 ranges from constitutively high in the brain where it is necessary for neuronal development and function, to inducible expression in haematopoietic and endothelial cells where it controls angiogenesis and immune activation. We show that expression of AGO2, a protein central to miRNA-mediated gene silencing and miRNA biogenesis, is negatively regulated by miR-132. Using HeLa cells, we demonstrate that miR-132 interacts with the AGO2 mRNA 3′UTR and suppresses AGO2 expression and AGO2-dependent small RNA-mediated silencing. Similarly, miR-132 over-expression leads to AGO2 suppression in primary human dermal lymphatic endothelial cells (HDLECs). During phorbol myristate acetate (PMA)-activation of HDLECs, miR-132 is induced in a CREB-dependent manner and inhibition of miR-132 results in increased AGO2 expression. In agreement with the role of AGO2 in maintenance of miRNA expression, AGO2 suppression by miR-132 affects the steady state levels of miR-221 and miR-146a, two miRNAs involved in angiogenesis and inflammation, respectively. Our data demonstrate that the miRNA-silencing machinery is subject to autoregulation during primary cell activation through direct suppression of AGO2 by miR-132.